Use of cell membrane penetrating indigoid bisindole derivatives

ABSTRACT

The present invention relates to the use of cell membrane penetrating indigoid bisindole derivatives for the manufacture of a medicament for the treatment of human solid cancers.

This application is a 371 of PCT/EP00/03210 filed Apr. 11, 2000.

The present invention relates to the use of cell membrane penetratingindigoid bisindole derivatives for the manufacture of a medicament forthe treatment of human solid cancers.

Indigoid bisindoles comprise a spectrum of natural dye stuffs. Many ofthese can be obtained from plants. Accordingly, indirubin, indigo andisoindigo are natural products which can be obtained from differentplants: namely, Baphicacanthus cusia (Acanthaceae), Indigoferasuffruticosa (Fabaceae), Isatis indigotica (Brassicaceae) and others.Indican, a glycoside which is found in plants, gives glucose and3-hydroxyindole due to acidic or enzymatic hydrolysis. 3-Hydroxy-indoleis converted by air-oxidation into indigo and its isomers. Indigonaturalis (Chinese: quingdai) is the natural blue dye obtained fromplant material, e.g. Isatis indigotica (Brassicaceae). Indirubin, anisomer of indigo, can be found in Indigo naturalis in an amount of up to60% (Falbe J. & Regitz M., Römpp Chemie Lexikon (1992), 9. Aufl.,Stuttgart, Georg Thieme Verlag). It occurs also in Isatis tinctoria inan amount of up to 5% which is indigenous to Central Europe (Gelius R.,Z. Chem., 20, (1980), 340-341). Derivatives of indirubin are known for along time as dyes of low persistence.

Indigo naturalis is reported to be used in traditional Chinese medicineas a haemostatic, anti-pyretic, anti-inflammatory and sedative agent inthe treatment of bacterial and viral infections. Antileukemic effects ofIndigo naturalis have also been reported, with indirubin being theeffective principle (Ji X. et al., Acta Pharm. Sin., 16, (1981),146-148; Gan W. J. et al., J. Hematol., 6, (1985), 611-613). In spite ofits anti-leukaemic activity, however, indirubin dissolves only poorly inwater and is therefore not readily resorbed. Recently, the antileukemicactivity of some better soluble indirubin derivatives has been reported(Ch. Li et a., Bull. Chem. Soc. Jpn. 69, 1621-1627 (1996)).

However, indigoid bisindole or its derivatives have never beeninvestigated with respect to solid tumors, in particular human solidtumors, and furthermore, the problem of the poor solubility resulting ina poor resorption has not been sufficiently solved yet.

Thus, the technical problem underlying the present invention is toprovide new active substances which can be used in the treatment ofhuman solid tumors and metastasis thereof. Furthermore, the resorptionof said substances should be improved in order to improve their in vivoanti-tumor activity.

The solution to the above technical problem is achieved by theembodiments characterized in the claims.

In particular, the present invention relates to the use of cell membranepenetrating indigoid bisindole derivatives for the manufacture of amedicament for the treatment of human solid tumors and metastasisthereof wherein the indigoid derivatives are selected from indigo,bis(3-phenylindol-2-yl), isoindigo and indirubin derivatives, the latterrepresented by the following formula (I):

wherein, when X represents an oxygen atom, R¹ represents a hydrogenatom, a halogen atom, a —NO₂ group, a methyl group, a sulfonamide groupor SO₂—NH—CH₂CH₂—OH; and

wherein, when X represents NOH, R¹ represents a hydrogen atom or aniodine atom.

The above indigoid bisindole derivatives can also be employed in theform of their physiologically acceptable salts. Furthermore, theindigoid bisindole derivatives according to the present invention mayalso be chemically coupled to masking agents as described e.g. in Germanpatent application DE-A-38 27 488 which function to carry the anti-tumoractive substances to the tumor.

In the following, the indigoid derivatives selected from indigo,isoindigo and indirubin derivatives according to the present inventionare also addressed to as “anti-tumor active compounds according to thepresent invention”.

According to the present invention the terms “cell membrane penetrating”and “cell resorbable” mean the ability of the indigoid bisindolederivatives to be taken up by the tumor cell through the cellularmembrane.

The term “human solid tumors” according to the present inventionpreferably includes carcinomas, melanomas, adenomas, sarcomas,lymphomas, neuroblastomas, teratomas, astrocytomas, glioblastomas andmesotheliomas. Specific examples are mammary carcinoma, large-cell lungcarcinoma, small-cell lung carcinoma, lung epidermoid andadenocarcinoma, colorectal carcinoma, bladder carcinoma, ovariancarcinoma, pancreatic carcinoma, renal carcinoma, prostatic carcinoma,head and neck carcinomas, melanomas, cervical carcinomas, osteosarcomaand the like.

The above identified indigoid bisindole derivatives of the presentinvention can be formulated into pharmaceutical compositions whichcontain optionally a pharmaceutically acceptable carrier and/or diluent.Said pharmaceutical compositions can be applied e.g. orally, topically,intravenously, intraperitoneally, subcutaneously and rectally inpharmaceutically effective amounts.

One general problem in the field of pharmacology is the formulation ofpharmaceutically active substances in pharmaceutical compositions whichcan be applied to a human body. Since most physiological fluids arewaterbased, the pharmaceutically active substances should be soluble inwater and/or a water mixable solvent wherein the latter of course has tobe physiologically acceptable in small concentrations, such as ethanol.Furthermore, pharmaceutically active substances which are taken orallyhave to be resorbed into surface of the human body—including thegastrointestinal mucous membrane—or, in case of an application viasyringe, e.g. intraperitoneal or intravasal, have to be resorbed throughthe cellular membranes of the of destination cells, specifically intothe tumor cells.

According to the present invention it has been found that in case of theindigoid bisindole derivatives according to the present invention, agood solubility is not the only prerequisite guaranteeing a goodanti-tumor activity in viva as it will become apparent by the Examplesand Comparative Examples shown below. An important factor for theanti-tumor activity of indigoid bisindole derivatives is their abilityto penetrate the cellular membranes of the tumor cells. Cellularmembranes are composed of lipids and compose a rather non-polar medium.Therefore, substitution with extremely polar groups such as thesulfonate group on the one hand improves the water solubility of acompound but on the other hand hinders or even prohibits the resorptionof anti-tumor active substances into a tumor cell. Thus, anti-tumoractive substances which show good anti-tumor activities under certain invitro conditions, have to be rejected because of not showing anyactivity when tested using intact cells or in vivo.

Therefore, in the following Examples the testing of the anti-tumoractive substances are tested by in vitro tests using intact tumor cellsand, additionally, in vivo tests. Furthermore, a comparison of theactivity test results and the tests evaluating the ability to penetratecellular membranes shows that indigoid bisindole compounds which exhibita good cell-penetrating ability also show good to excellent anti-tumoractivity.

THE FIGURES SHOW:

FIG. 1 is a graph which shows the development of the relative tumorvolume with time during chemotherapy of LXFL 529/17 with indigoidbisindole derivatives according to the present invention (compoundsaccording to Examples 1, 4 and 6). The anti-tumor active substancesaccording to the present invention were applied intraperitoneally tonude mice in doses and according to the schedule as described below inTable 4. Compared to the vehicle control, all compounds significantlyinhibited the tumor growth.

FIG. 2 is a graph which shows the relative body weight change of thetested nude mice with time during chemotherapy of LXFL 529/17.5-Methylindirubin (Example 6) at a dosage of 100 mg/kg up to 300 mg/kgshowed very high anti-tumor activity (FIG. 1 and FIG. 3) without anysignificant reduction of body weight (FIG. 2 and FIG. 4) thusdemonstrating high anti-tumor activity without significant toxicity.

FIG. 3, FIG. 5 and FIG. 7 are graphs which show the relative tumorvolume versus the time during the chemotherapy of LXFL 529/17 with otherindigoid bisindole derivatives according to the present invention(compounds according to Examples 8, 9, 10 and 14).

FIG. 4, FIG. 6 and FIG. 8 are graphs which show the relative body weightchange of the tested nude mice with time during chemotherapy of LXFL529/17 using said other indigoid bisindole derivatives according to thepresent invention.

The present invention is explained in detail by the following examplesand comparative examples by which also further advantages of the presentinvention will become apparent.

1. SYNTHESIS OF THE INDIGOID BISINDOLE DERIVATIVES EXAMPLE 1 Indirubin

To a solution of 0.42 g (2.4 mmol) of indoxyl acetate in 20 ml methanolunder argon 0.35 g (2.4 mmol) of satin and 0.55 g (5.2 mmol) of sodiumcarbonate are added. The mixture is stirred for 30 min at ambienttemperature. After 24 h standing at ambient temperature, the reactionmixture is filtered off. The precipitate is washed with little methanoland water until the filtrate shows a neutral pH. Residual water isremoved by storage in an evacuated exsiccator over potassium hydroxide.Recrystallisation from ethanol or pyridine gives deep purple crystals(Russell G. A., Kaupp G. (1969), J. Am. Chem. Soc., 91, 3851-9,modified).

Yield: 0.51 g (81%), fine, deep-purple needles, Fp: 341-343° C.;CHN-analysis: (C₁₆H₁₀N₂O₂); MW: 262.26 g/mol; calc.: 73.3% C, 3.8% H,10.7% N; found: 73.2% C, 4.0% H, 10.6% N; mass spectrum: m/z=262: (M⁺,100%), 234: (43%), 205 (25%), 158 (3%), 131 (4%), 103 (7%), 76 (3%);¹H-NMR and ¹³C-NMR-spectrum are in accordance with the proposedstructure. IR-spectrum: 3340cm⁻¹: v (N—H), 1710 cm⁻¹: v (3′—C═O), 1650cm⁻¹: v (2—C═O), 1590 cm⁻¹: v (C═C, aryl), 1450 cm⁻¹: v (C═C, aryl), 745cm⁻¹: v (aryl with four neighbouring H-atoms). UV/Vis-spectrum (DMSO):290 nm, 363 nm, 383 nm (shoulder), 551 nm.

Essentially the same synthetic procedure was applied for the followingExamples 2 to 9, 12, 13 and Comparative Examples 1 and 2:

EXAMPLE 2 5-Lodoindirubine

Yield: 80%, fine, deep-purple needles, Fp: 334-335° C. (decomposition);CHN-analysis (C₁₆H₉IN₂O₂); MG=388.16 g/mol; calc.: 49.5% C, 2.3% H, 7.2%N; found.: 49.7% C, 2.5% H, 7.1% N; Mass spectrum: 388 (M⁺, 100%), 360(3%), 269 (9%), 261 (6%), 233 (16%), 205 (16%), 128 (1%); ¹H-NMR- and¹³C-NMR-spectrum are in accordance with the proposed structure.UV/Vis-spectrum (DMSO): 370 nm, 386 nm (shoulder), 555 nm.

EXAMPLE 3 5-Bromoindirubin

Yield: 70%, fine, deep-purple needles; CHN-analysis (C₁₆H₉BrN₂O₂);MG=341.16 g/mol, calc.: 56.3% C, 2.7% H, 8.2% N; found 56.4% C, 2.7% H,8.2% N; Mass spectrum: 342(M⁺, 100%), 340 (M⁺, 99%), 314 (18%), 262(64%), 233 (34%), 205 (81%), 177 (10%); ¹H-NMR- and ¹³C-NMR-spectrum arein accordance with the proposed structure.

EXAMPLE 4 5-Chloroindirubin

Yield: 95%, fine, deep-purple needles; CHN-analysis (C₁₆H₉CIN₂O₂);MG=296.70 g/mol; calc.: 49.5% C, 2.3% H, 7.2% N; found: 49.7% C, 2.5% H,7.1 % N; Mass spectrum: m/z=296 (M⁺, 100%), 268 (39%), 239 (8%), 233(35%), 205 (50%), 177 (7%), 153 (6%), 137 (7%), 77 (7%), 120 (4%), 102(6%), 77 (7%). ¹H-NMR- and ¹³C-NMR-spectrum are in accordance with theproposed structure.

EXAMPLE 5 5-Fluoroindirubin

Yield: 92%, fine, deep-purple needles; CHN-analysis (C₁₆H₉FN₂O₂),MG=280.25 g/mol, calc.: 68.6% C, 3.2% H, 9.9% N; found: 68.0% C, 3.2% H,9.9% N; Mass spectrum: m/z=281 (M⁺+H⁺, 19%), 280 (M⁺, 100%), 252 (73%),223 (32%), 176 (6%), 140 (7%), 121 (13%), 94 (4%), 76 (12%), 77 (7%), 57(4%), 44(15%). ¹H-NMR- and ¹³C-NMR-spectrum are in accordance with theproposed structure.

EXAMPLE 6 5-Methylindirubin

Yield: 92%, fine, deep-purple needles; CHN-analysis (C₁₇H₁₂N₂O₂),MG=276.28 g/mol, calc.: 73.9% C, 4.4% H, 10.1% N; found: 73.8% C, 4.3%H, 10.2% N; Mass spectrum: m/z=276 (M⁺, 100%), 261 (10%), 248 (47%), 247(53%), 220 (6%), 219 (18%), 205 (7%), 171 (4%), 165 (10%), 138 (4%), 133(15%), 104 (7%), 77 (7%); ¹H-NMR- and ¹³C-NMR-spectrum are in accordancewith the proposed structure.

EXAMPLE 7 5-Nitroindirubin

Yield: 88%, fine, deep-purple needles; CHN-analysis (C₁₆H₉N₃O₄),MG=307.26 g/mol; calc.: 62.5% C, 3.0% H, 13.7% N; found: 62.4% C, 3.0%H, 13.3% N; Mass spectrum: m/z=307 (M⁺, 5%), 276 (10%), 262 (100%), 234(23%), 205 (22%), 158 (6%), 131 (10), 104 (19%), 76 (12%), 50 (6%).¹H-NMR- and ¹³C-NMR-spectrum are in accordance with the proposedstructure.

EXAMPLE 8 Indirubin-3′-oxime

Indirubin-3′-oxime was synthesized by reaction of indirubin withhydroxylamine hydrochloride in a pyridine solution (Farbwerke vorm.Meister Lucius & Brüning in Hoechst a.M., Patentschrift desReichspatentamtes Nr. 283726 (1913)). ¹³C-NMR-spectroscopy revealed thelocation of the hydroxyimino residue in 3′-Position (δ(C2)=171.05 ppm;δ(C3′)=145.42 ppm; DMSO-d₆, RT).

Yield: 90%, red crystals; CHN-analysis (C₁₆H₁₁N₃O₂), MG=277.30 g/mol;calc.: 69.3% C, 4.0% H, 15.2% N; found: 69.0% C, 4.0% H, 14.9% N;¹H-NMR- and ¹³C-NMR-spectrum are in accordance with the proposedstructure.

EXAMPLE 9 5-Lodoindirubine-3′-oxime

Indirubin-3′-oxime was synthesized by reaction of 5-lodoindirubine withhydroxylamine hydrochloride in a pyridine solution. ¹³C-NMR-spectroscopyrevealed the location of the hydroxyimino residue in 3′-Position(δ(C2)=170.25 ppm; δ(C3′)=151.52 ppm; DMSO-d₆, RT).

Yield: 90 %, red crystals; CHN-analysis (C₁₆H₁₀IN₃O₂), MG=403,20 g/mol;calc.: 47.7% C, 2.5% H, 10.4% N; found: 47.1% C, 2.5% H, 10.1% N;¹H-NMR- and ¹³C-NMR-spectrum are in accordance with the proposedstructure.

EXAMPLE 10 Isoindigo

Isoindigo was synthesized by reaction of oxindole with isatin in aceticacid with addition of hydrochloric acid (Wahl A., Bayard P., ComptesRendues Hebdomadaires des Seances de L'Academie des Sciences, 148,(1909), 716-719).

Yield: 84%, crystalline, brown substance; CHN-analysis (C₁₆H₁₀N₂O₂),MG=262.26 g/mol; calc.: 73.3% C, 3.8% H, 10.7% N; found: 73.0% C, 3.8%H, 10.9% N; Mass spectrum: m/z=262 (M⁺, 100%), 234 (85%), 220 (5%), 205(18%), 190 (4%), 177 (5%), 151 (5%), 132 (17%), 103 (6%), 76 (4%), 32(26%). ¹H-NMR- and ¹³C-NMR-spectrum are in accordance with the proposedstructure.

EXAMPLE 11 Indigo

Chemical grade indigo was purchased by Fluka Chemie AG.

EXAMPLE 12 Indirubin-5-sulfonamide

¹H-NMR- and ¹³C-NMR-spectrum are in accordance with the proposedstructure.

EXAMPLE 13 Indirubin-5-Sulfone(2-hydroxyethyl)amide

¹H-NMR- and ¹³C-NMR-spectrum are in accordance with the proposedstructure.

EXAMPLE 14 Bis(3-Phenylindol-2-yl

To a cooled solution of 2-aminobenzophenone in dichloromethane andpyridine under inert gas, a solution of oxalyl chloride indichloromethane is dropped. After completion of the reaction, 0.5 nhydrochloric acid is added, the formed precipitate is filtrated off andwashed subsequently with 0.5 n hydrochloric acid, a solution of sodiumhydrogencarbonate and water. The obtained product(N,N′-bis(2-benzoylphenyl)-oxamide), zinc dust and titanium(III)chlorideare suspended in dimethoxyethane and heated to reflux. After heating for3 h, the mixture is cooled to ambient temperature and the precipitate isfiltrated off and washed with ethyl acetate. The crude product ispurified using column chromatography (silica gel), then dissolved inethyl acetate and precipitated in form of white crystals by addingpetrol ether.

CHN-analysis (C₂₈H₂₀N₂), MG=384.48 g/mol; calc.: 87.5% C, 5.2% H, 7.3%N; found: 87.3% C, 5.3% H, 7.3% N; ¹H-NMR- and ¹³C-NMR-spectrum are inaccordance with the proposed structure.

COMPARATIVE EXAMPLE 1 Indirubin-5-sulfonic Acid

Yield: 76%, crystalline, deep-purple substance; Mass spectrum: 388 (M⁺,100%), 360 (3%), 269 (9%), 261 (6%), 233 (16%), 205 (16%), 128 (1%).¹H-NMR- and ¹³C-NMR-spectrum are in accordance with the proposedstructure.

COMPARATIVE EXAMPLE 2 Indirubin-3′-oxime-5-sulfonic acid

Yield: 76%, crystalline, deep-purple substance; Mass spectrum: 388 (M⁺,100%), 360 (3%), 269 (9%), 261 (6%), 233 (16%), 205 (16%), 128 (1%).¹H-NMR- and ¹³C-NMR-spectrum are in accordance with the proposedstructure.

Table 2 summarizes the structures of the indirubin compounds of Examples1 to 9 and Comparative Examples 1 and 2.

TABLE 1

compound R¹ X Example 1 Indirubin H O 2 5-Iodoindirubin I O 35-Bromoindirubin Br O 4 5-Chloroindirubin Cl O 5 5-Fluoroindirubin F O 65-Methylindirubin CH₃ O 7 5-Nitroindirubin NO₂ O 8 Indirubin-3′-oxime HNOH 9 5-Iodoindirubine-3′-oxime I NOH 10  Isoindigo 11  Indigo 12 Indirubin-5-sulfonamide SO₂—NH₂ O 13 Indirubin-5-sulfone(2-hydroxyethyl)amide SO₂—NH—CH₂CH₂OH O 14 Bis(3-phenylindol-2-yl) Comparative Examples 1 Indirubin-5-sulfonic acidSO₃H O 2 Indirubin-3′-oxime-5-sulfonic acid SO₃H NOH

2. CELLULAR UPTAKE INTO LXFL 529L CELLS

The compounds of Examples 1, 6 and 8 and Comparative Examples 1 and 2were investigated with respect to their ability to penetrate LXFL 529Lcells having the passage numbers P23 to P39. The results are shown inTable 2. The amounts of the substances taken up by the cells are givendepending on the concentration of the substance within the incubationmedium. The time of incubation was 2 hours in all of the experiments.Furthermore, the distribution of the substance which was taken up by thecells in the cytosol and the cellular organelles (particular) wasestimated and is given in the intermediate column of Table 2. Tumor cellgrowth inhibition was determined by the sulfo-rhodamine B assay (SRBassay) according to Skehan et al., J. Natl. Cancer Institute 82, pages1107-1112 (1990). Incubation was conducted for three days in serumcontaining medium. Tumor cell lines tested were a large-cell lungcarcinoma xenograft line LXFL 529 L and the mammary carcinoma lineMCF-7. Results are given as IC₅₀ [μM] corresponding to the concentrationof compounds inducing 50% growth inhibition, compared to vehicle treatedcontrol.

TABLE 2 amount of substance Tumor cell growth concentra- within thedistribution inhibition (SRB-assay) tion of cells [%] IC₅₀ incubation[μm/mg cellular [μM] substance [μM] protein] cytosol organelles LXFL529LMCF7 Example 1 10 0.15 ± 0.08  7 ± 5.7 93 ± 5.7 9.9 ± 0.1 4.0 ± 2.0 200.20 ± 0.08  6 ± 1.4 94 ± 1.4 Example 6 10 0.52 ± 0.1  13 ± 0.7 87 ± 0.77.5 ± 0.5 4.8 ± 0.5 20 0.86 ± 0.22  6 ± 2.8 94 ± 2.8 Example 8 10 0.16 ±0.01 43 ± 14.1 57 ± 14.1 3.0 ± 0.5 3.3 ± 0.4 20 0.23 ± 0.03 44 ± 15.6 56± 15.6 Comparative 10 <0.02  —  — >100 >100 Example 1 20 <0.02  —  —Comparative 10 <0.05  —  — >100 >100 Example 2 20 <0.05  —  —

The compounds according to the Examples 1, 6 and 8 were all taken up bythe tumor cells. The ability of the compound according to Example 6 topenetrate the cellular membrane is substantially improved compared tothat of the parent compound indirubin (Example 1). The uptake of thecompound according to Example 8 is also slightly improved compared tothe non-substituted indirubin (Example 1).

The compounds of Comparative Examples 1 and 2 were essentially not takenup by the cells although these compound are well soluble inphysiological solutions. Obviously, the sulfonate group hinders thepenetration through the cellular membrane. Furthermore, referring toComparative Example 2, this detrimental effect cannot be compensated bythe introduction of an oxime group.

3. EVALUATION OF THE ANTI-TUMOR ACTIVITY

The anti-tumor activity of the compounds was evaluated via acolony-forming-assay as described e.g. by D. P. Berger et al. in Annalsof Oncology 1, pages 333-341 (1 990), “The clonongenic assay with humantumor xenografts, evaluation, predictive values and application for drugscreening”.

The experiments were conducted using various tumor cell lines, inparticular mammary carcinoma (MAXF), lung adenocarcinoma (LXFA),large-cell lung carcinoma (LXFL), small-cell lung carcinoma (LXFS),colon carcinoma (CXF), melanoma (MEXF), pancreatic carcinoma (PAXF),renal carcinoma (RXF), ovarian carcinoma (OVXF) and bladder carcinoma(BXF).

The IC₇₀-values and IC₅₀-values, respectively, define the concentrationof a pharmaceutically active compound causing 70% and 50%, respectively,reduction of colony formation compared to the untreated control.Therefore, IC₇₀- and IC50-values serve to demonstrate the anti-tumoractivity of a pharmaceutically active compound wherein low IC₇₀- and/orIC₅₀-values demonstrate a superior anti-tumor activity. According to thepresent invention, the IC₇₀-value preferably is 20 μM or lower, morepreferably 10 μM or lower.

Table 3 shows the anti-tumor activity of the compounds according to theExamples and Comparative Example 1. The compounds according to theinventive Examples show good to excellent anti-tumor activity againstvarious types of tumor cell lines. The compound according to ComparativeExample 1 does not exhibit an anti-tumor activity against any of thetumor lines. This behavior is in accordance with the lacking ability ofthis substance to penetrate cellular membranes as demonstrated in Table2, above.

Surprisingly, small variations in the substitution pattern result inremarkable changes in the anti-tumor activity profile. However, almostall compounds according to the Examples exhibit good anti-tumor activityagainst mammary carcinoma.

TABLE 3 IC₅₀ IC₇₀ tumor xenograft Example [μM] [μM] type xenograft  1(indirubin) 25.3 35.6 lung large-cell LXFL529 2.0 6.0 mammary MCF7X12.3 >30 ovarian OVXF1353 5.4 >30 pancreatic PAXF736  2(5-iodo-indirubin) 6.3 >30 colon HT29X 8.0 23 lung adeno carcinomaLXFA526 13.7 24.5 lung small-cell LXFS650 <1.0 2.5 mammary MCF7X18.0 >30 pancreatic PAXF546  3 (5-bromo-indirubin) <1.0 17.3 colon HT29X2.3 14.4 lung adenocarcinoma LXFA526 <1.0 <1.0 mammary MCF7X 3.4 8.0melanoma MEXF514 13.2 >30 pancreatic HT29X 04 (5-chloro-indirubin) <1.0<1.0 mammary MCF7X 17.1 26.0 melanoma MEXF514 3.2 8.0 pancreatic PAXF73611.2 24.7 renal 1220 4.6 >30 pancreatic PAXF546 <1 17.3 colon HT29X  5(5-fluoro-indirubin) <1.0 <1.0 mammary MCF7X <1.0 6.1 ovarian OVXF1353<1.0 1.1 pancreatic PAXF736  6 (5-methyl-indirubin) <1.0 14.4 colonHT29X <1.0 1.2 mammary MCF7X 19.2 27.8 melanoma MEXF514 15.4 27.5pancreatic PAXF736 1.0 >30 ovarian OVXF1352  7 (5-nitro-indirubin) <1.015.1 mammary MCF7X 4.9 >10.0 melanoma MEXF514  8 (indirubin-3′-oxime)10.6 16.1 bladder BXF1301 8.0 12.6 colon CXF280 0.9 3.4 lungadenocarcinoma LXFA289 7.7 9.2 mammary MX1 1.0 2.6 melanoma MEXF989 2.85.7 melanoma MEXF515LX  9 (5-iodo-3′-oxime-indirubin) 4.0 5.8 bladderBXF1301 10.7 16.3 colon CXF280 0.05 0.7 lung adenocarcinoma LXFA289 2.410.4 mammary MCF7X 2.6 4.9 melanoma MEXF515LX 10 (isoindigo) 6.0 8.2bladder BXF1301 <1.0 <1.0 colon CXF280 2.6 4.5 lung large-cell LXFL529<1.0 <1.0 lung small cell LXFS650 <1.0 <1.0 mammary MX1 <1.0 <1.0mammary MCF7X <1.0 <1.0 melanoma MEXF989 <1.0 <1.0 ovarian OVXF1355 <1.0<1.0 pancreatic PAXF546 <1.0 <1.0 pancreatic PAXF736 <1.0 <1.0 colonHT29X 11 (indigo) 3.3 26.1 colon HT29X 3.5 12.3 lung adenocarcinomaLXFA289 3.9 16.7 ovarian OVXF1353 13 (5-SO₂—NH—CH₂—CH₂—OH-indirubin)12.0 17.3 colon CXF280 1.1 2.6 lung adenocarcinoma LXFA289 3.4 5.9 lunglarge-cell LXFL529 0.6 2.1 mammary MCF7X <0.1 0.4 melanoma MEXF515LX 0.30.4 ovarian OVXF899 14 (Bis(3-phenylindol-2-yl)) 2.7 6.2 bladder BXF1299<1.0 7.2 colon CXF280 1.6 3.4 colon HT29X 4.7 6.7 lung small-cellLXFS650 2.8 4.8 mammary MX1 Comp. 1 >30.0 >30.0 (all)

4. IN VIVO EXPERIMENTS

Compound of Examples 1, 4, 6, 8, 9 and 10 were subjected to in vivotesting in nude mice bearing subcutaneously growing human tumorxenograft LXFL 529. The indigoid bisindole derivatives were appliedintraperitoneally to the animals in doses and according to the scheduleas described in Table 4.

TABLE 4 doses schedule of application activity Graph shown [mg/kg/day][day(s)] rating in Figure Ex. 1 100 1-5, 8-9 + FIG. 1 and 2 200 1-5, 8-9++ FIG. 1 and 2 Ex. 4 100 1-5 ++ FIG. 1 and 2 300 1-5 ++ FIG. 1 and 2Ex. 6 100 1-5, 8-12, 15, 17, 19, 22 ++ FIG. 1 and 2 300 1, 4, 8, 11, 15,18, 22 ++ FIG. 3 and 4 Ex. 8 100 1, 4, 8, 11, 15, 18, 22 − FIG. 3 and 4300 1, 4, 8, 11, 15, 18, 22 − FIG. 3 and 4 Ex. 9 100 1, 4, 8, 11, 15,18, 22 − FIG. 3 and 4 300 1, 4, 8, 11, 15, 18, 22 + FIG. 3 and 4 Ex. 10 30 1-5 − FIG. 5 and 6 100 1-5 − FIG. 5 and 6 300 1-5 + FIG. 5 and 6 Ex.14  10 1-5, 8-12 − FIG. 7 and 8 100 1-5, 8-12 + FIG. 7 and 8 300 1-5,8-12 + FIG. 7 and 8

The experiments were run for 21 or 28 days. Anti-tumor activity wasevaluated comparing the median tumor volume relative to control,expressed as %T/C, wherein T is the test group and C the vehicle controlgroup. In Table 4, anti-tumor activity is given according to an activityrate scale.

Activity rating: − inactive T/C > 50% + tumor inhibition T/C > 25-50% ++tumor stasis T/C ≦ 25%

The result are further demonstrated by FIGS. 1 to 8.

A reduction of the body weight of the tested mice of more then 20% byweight in general is interpreted as a toxic dose.

What is claimed is:
 1. A method for the treatment of human solid tumorsand metastasis thereof comprising, administering a cell membranepenetrating indigoid bisindole derivative to a human in need thereof,wherein said human solid tumors and metastasis thereof are selected fromcarcinomas, melanomas, adenomas, sarcomas, lymphomas, neuroblastomas,teratomas, astrocytomas, glioblastomas, or mesotheliomas, and theindigoid bisindole derivative is selected from bis(3-phenylindol-2-yl),isoindigo or indirubin derivatives, the indirubin derivatives beingrepresented by the following formula (I):

wherein, X represents an oxygen atom or NOH, and when X represents anoxygen atom, R¹ represents a hydrogen atom, a halogen atom, a —NO₂group, a methyl group, a sulfonamide group or SO₂—NH—CH₂CH₂—OH; and whenX represents NOH, R¹ represents a hydrogen atom or an iodide atom. 2.The method according to claim 1, wherein the solid tumors are selectedfrom mammary carcinoma, melanoma, large-cell lung carcinoma, small-celllung carcinoma, lung epidermoid and adenocarcinoma, colorectalcarcinoma, bladder carcinoma, ovarian carcinoma, pancreatic carcinoma,renal carcinoma, prostatic carcinoma, head and neck carcinomas,melanomas, cervical carcinomas, or osteosarcoma.
 3. The method accordingto claim 1, wherein the indigoid bisindole derivative is in the form ofa physiologically acceptable salt.
 4. The method according to claim 2,wherein the indigoid bisindole derivative is in the form of aphysiologically acceptable salt.
 5. The method according to claim 1,wherein the solid tumors are selected from mammary carcinoma, melanoma,large-cell lung carcinoma, small-cell lung carcinoma, lungadenocarcinoma, colon carcinoma, bladder carcinoma, ovarian carcinoma,pancreatic carcinoma, renal carcinoma, or prostatic carcinoma.